1. Field of the Invention
The present invention relates to novel chemical syntheses of methionylmethionine, the dipeptide of methionine, and the specific use thereof as feed additive alone or mixed with methionine for fish and crustacean nutrition.
2. Description of the Related Art
Essential amino acids (EAA) such as methionine, lysine or threonine are very important constituents as feed additives in animal nutrition and play a significant part in the commercial rearing of productive animals such as, for example, chickens, pigs and ruminants. Supplementation of natural protein sources such as, for example, soybeans, corn and wheat with EAAs makes it possible on the one hand for the animals to grow faster, or for milk production to be higher in high-output dairy cows, but on the other hand for the utilization of the feed to be more efficient. This represents a very great commercial advantage. The markets for feed additives are of great industrial and commercial importance. In addition, they are high-growth markets, attributable not least to the increasing importance of countries such as, for example, China and India.
L-Methionine ((S)-2-amino-4-methylthiobutyric acid) represents the first limiting amino acid for many species such as chickens, ducks, turkeys and also for many fish and shellfish species and therefore plays a very significant part in animal nutrition and as feed additive (Rosenberg et al., J. Agr. Food Chem. 1957, 5, 694-700 and Lovell, T. R., J. Anim. Sci. 1991, 69, 4193-4200). However, in the classical chemical synthesis, methionine results as racemate, a 50:50 mixture of D- and L-methionine. This racemic DL-methionine can, however, be employed directly as feed additive because there is in some species under in vivo conditions a transformation mechanism which converts the unnatural D enantiomer of methionine into the natural L enantiomer. This entails firstly the D-methionine being deaminated with the aid of a nonspecific D-oxidase to α-ketomethionine, and subsequently being further transformed with an L-transaminase into L-methionine (Baker, D. H. in “Amino acids in farm animal nutrition”, D'Mello, J. P. F. (ed.), Wallingford (UK), CAB International, 1994, 37-61). The available amount of L-methionine in the body is increased thereby and can then be available to the animal for growth. The enzymatic transformation of D- to L-methionine has been detected in chickens, pigs and cows, but especially also in carnivorous and omnivorous fish and also in shrimps and prawns. Thus, for example, Sveier et al. (Aquacult. Nutr. 2001, 7 (3), 169-181) and Kim et al. (Aquaculture 1992, 101 (1-2), 95-103) were able to show that the transformation of D- into L-methionine is possible in carnivorous Atlantic salmon and rainbow trout. Robinson et al. (J. Nutr. 1978, 108 (12), 1932-1936) and Schwarz et al. (Aquaculture 1998, 161, 121-129) were able to show the same for omnivorous fish species such as; for example, catfish and carp. In addition, Forster and Dominy (J. World Aquacult. Soc. 2006, 37 (4), 474-480) were able to show in feeding experiments on omnivorous shrimps of the species Litopenaeus vannamei that DL-methionine has the same activity as L-methionine.
The world production in 2007 of crystalline DL-methionine and racemic, liquid methionine hydroxy analog (MHA, rac-2-hydroxy-4-(methylthio)butanoic acid (HMB)) and solid calcium MHA was more than 700 000 t, which was successfully employed directly as feed additive for monogastric animals such as, for example, poultry and pigs. Owing to the rapid commercial development of fish and crustacean farming in highly industrialized aquacultures an optimal, economical and efficient methionine supplementation option has become increasingly important precisely in this area in recent years (Food and Agriculture Organization of the United Nation (FAO) Fisheries Department “State of World Aquaculture 2006”, 2006, Rome, International Food Policy Research Institute (IFPRI) “Fish 2020: Supply and Demand in Changing Markets”, 2003, Washington, D.C.). However, in contrast to chickens and pigs, various problems occur on use of methionine, MHA or Ca-MHA as feed additive for certain fish and crustacean varieties. Thus, Rumsey and Ketola (J. Fish. Res. Bd. Can. 1975, 32, 422-426) report that the use of soybean meal in conjunction with singly supplemented crystalline amino acids did not lead to any increase in growth of rainbow trout. Murai et al. (Bull. Japan. Soc. Sci. Fish. 1984, 50, (11), 1957) were able to show that daily feeding of fish diets with high rates of supplemented crystalline amino acids in carp led to more than 40% of the free amino acids being excreted via the gills and kidneys. Because of the rapid absorption of supplemented amino acids shortly after feed intake, there is a very rapid rise in the amino acid concentration in the fish's blood plasma (fast response). However, at this time, the other amino acids from the natural protein sources such as, for example, soybean meal are not yet present in the plasma, possibly leading to asynchronicity of the concurrent availability of all the important amino acids. As a result thereof, part of the highly concentrated amino acids is rapidly excreted or rapidly metabolized in the body, and is used for example as pure energy source. As a result, there is only a slight or no increase in growth, upon use of crystalline amino acids as feed additives (Aoe et al., Bull. Jap. Carp Soc. Sci. Fish. 1970, 36, 407-413). Supplementation of crystalline amino acids may lead to further problems in crustaceans. The slow feeding behavior of certain crustaceans such as, for example, shrimps of the species Litopenaeus Vannamei results, owing to the long residence time of the feed under water, in the supplemented, water-soluble amino acids being dissolved out (leaching), leading to eutrophication of the water and not to an increase in growth of the animals (Alam et al., Aquaculture 2005, 248, 13-16).
Efficiently supplying fish and crustaceans kept in aquacultures thus requires, for certain species and applications, a specific methionine product form, such as, for example, an appropriately chemically or physically protected methionine. The aim of this is on the one hand that the product remains sufficiently stable in the aqueous environment during feeding and is not dissolved out of the feed. On the other hand that the methionine product eventually taken in by the animal can be utilized optimally and with high efficiency in the animal body.
Many efforts have been made in the past to develop suitable feed additives, particularly based on methionine, for fish and crustaceans. Thus, for example, WO8906497 describes the use of di- and tripeptides as feed additive for fish and crustaceans. The intention of this is to promote the growth of the animals. However, the di- and tripeptides preferably employed in this case were from nonessential and therefore also nonlimiting amino acids such as, for example, glycine, alanine and serine. The only methionine-containing dipeptides described are DL-alanyl-DL-methionine and DL-methionyl-DL-glycine. However, this means that effectively only 50% of active substance (mol/mol) are present in the dipeptide, and this must be categorized as very disadvantageous from the aspect of economics. WO02088667 describes the enantioselective synthesis and use of oligomers of MHA and amino acids such as, for example, methionine as feed additives, inter alia also for fish and crustaceans. It is said to be possible to achieve faster growth thereby. The described oligomers are assembled by an enzyme-catalyzed reaction and exhibit a very broad distribution of the chain lengths of the individual oligomers. This makes the process unselective, costly and elaborate in the procedure and purification. Dabrowski et al. describes in US20030099689 the use of synthetic peptides as feed additives for promoting the growth of aquatic animals. In this case, the proportion of the peptides in the complete feed formulation may be 6-50% by weight. The synthetic peptides preferably consist of essential and limiting amino acids. However, the synthesis of such synthesized oligo- and polypeptides is very elaborate, costly and difficult to convert to the industrial scale. In addition, the effectiveness of polypeptides of a single amino acid is disputed, because these are often converted only very slowly or not at all under physiological conditions into free amino acids. Thus, for example, Baker et al. (J. Nutr. 1982, 112, 1130-1132) describes the lack of biological value of poly-L-methionine in chickens because of the absolute insolubility in water, since absorption by the body is impossible.
Besides the use of novel chemical methionine derivatives such as, for example, methionine-containing peptides and oligomers, there has also been investigation of various physical protection possibilities such as, for example, coatings and the incorporation of an amino acid in a protective matrix. Thus, for example, Alam et al. (Aquacult. Nutr. 2004, 10, 309-316 and Aquaculture 2005, 248, 13-19) were able to show that coated methionine and lysine has, in contrast to uncoated, a very positive influence on the growth of young kuruma shrimps. Although use of a specific coating was able to suppress the leaching of methionine and lysine out of the feed pellet, there are some serious disadvantages. The preparation or the coating of methionine usually represents a technically complicated and elaborate process and is therefore costly. In addition, the surface coating of the methionine after coating is easily damaged by mechanical stress and abrasion during feed processing, possibly leading to a diminution or complete loss of the physical protection. An additional factor is that the content of methionine is reduced, and thus often becomes uneconomic, by a coating or use of a matrix substance.
Besides the inventive novel use of DL-methionyl-DL-methionine as feed additive with low leaching characteristics from feed pellets and extrudates, and an optimal supply of methionine to the body through slow-release cleavage of methionylmethionine, it has also been possible to develop novel processes for preparing methionylmethionine which have many advantages over the preparation variants described in the literature. Most of the dipeptide syntheses disclosed in the literature use costly protective groups such as, for example, Boc-(tert-butoxycarbonyl) or Z-(benzyloxycarbonyl) protective groups, which have to be attached to the appropriate amino acid before the actual dipeptide synthesis, and subsequently eliminated again. In addition, activation of the amino acids to be coupled is usually necessary. Thus, methionylmethionine can be prepared by coupling N-Boc-methionine with the methyl ester of methionine using dicyclohexylcarbodiimide (DCC). The great disadvantages of this preparation process are the use of costly protective groups, a very elaborate synthesis and costly coupling reagents which cannot be recycled, such as, for example, DCC. Another alternative for the industrial synthesis of methionylmethionine is described in DE2261926. 3,6-Bis[2-methylthio)ethyl]-2,5-piperazinedione (methioninediketopiperazine, DKP) is formed in the first stage by heating the isopropyl ester of methionine and is then hydrolyzed to methionylmethionine. Merely satisfactory yields of 62-65% were possible for the hydrolysis step in this case. In addition, the use of methionine isopropyl ester as starting material is too costly and therefore uneconomic.